Angiogenesis is an essential component of normal processes such as growth and tissue repair (Conway, E. M., et al. Cardiovasc Res 49:507-21. (2001)) (Folkman, J. Semin Oncol 28:536-42. (2001)). Angiogenesis is also involved in the development of many pathological situations commonly called angiogenic diseases. Examples of pathological conditions leading to angiogenic diseases are, among other, arthritis, diabetic retinopathy, psoriasis, obesity and cancer (Carmeliet, P. Nat Med 9:653-60 (2003)). In the latter case, formation of new capillaries is essential not only for the growth of the primary tumor but also for the spreading of metastases (Fidler, I. J. Nat Rev Cancer 3:453-8 (2003)).
Recently the search for angiogenic inhibitors has been vigorously pursued. Currently, several angiogenic inhibitors including thrombospondin, platelet factor 4, angiostatin, endostatin, fumagillin and thalidomide are being studied. Several of these are in clinical trials e.g. fumagillin and thalidomide.
Also recently a new antiangiogenic factor has been identified: the 16 kDa N-terminal fragment of prolactin (human 16K prolactin or 16K hPRL). It was shown that 16K hPRL displays antiangiogenic properties both in vitro and in vivo (Ferrara, N., et al. Endocrinology 129:896-900 (1991), Clapp, C., et al. Endocrinology 133:1292-9. (1993), Lee, H., et al. Endocrinology 139:3696-703 (1998)). The 16K hPRL inhibits endothelial cell proliferation and induces their apoptosis (D'Angelo, G., et al. Proc Natl Acad Sci USA 92:6374-8 (1995), D'Angelo, G., et al. Mol Endocrinol 13:692-704 (1999), Martini, J. F., et al. Mol Endocrinol 14:1536-49. (2000)). Indeed, treatment of BEC (brain endothelial cell) with recombinant 16K prolactin increases DNA fragmentation in BEC in a time- and dose-dependent fashion (Martini, J. F., et al. Mol Endocrinol 14:1536-49. (2000)). 16K prolactin-induced apoptosis is correlated with the rapid activation of caspases 1 and 3 and an increase in the conversion of Bcl-X to its proapoptotic form. Further it was determined that the NF-κB signaling pathway is involved in mediating the apoptotic action of 16K hPRL in BEC. In a dose-dependent manner, treatment with 16K hPRL induces IκB-α degradation permitting translocation of NF-κB to the nucleus and reporter gene activation. Inhibition of NF-κB activation by overexpression of a non-degradable IκB-α mutant or treatment with NF-κB inhibitors blocks 16K hPRL-induced apoptosis. Treatment with 16K hPRL activates the initiator caspases 8 and 9 and the effector caspase 3, all of which are essential for inducing endothelial cell apoptosis. Activation of the caspases cascade by 16K hPRL is also NF-κB-dependent (Tabruyn, S. P., et al. Mol Endocrinol 17:1815-23 (2003)). 16K hPRL was shown to inhibit proliferation by inducing a cell cycle arrest at both the G1-S and G2-M phases (Tabruyn et al., Mol Endocrinol, 19, 1932-1942 (2005)).
In a recent publication it was disclosed that inhibition of angiogenesis by 16K hPRL is able to prevent the growth of subcutaneously implanted human colon tumors in the mouse (Bentzien, F., et al. Cancer Res 61:7356-62. (2001)). More recently, an adenovirus vector was engineered allowing the expression of 16K hPRL in situ. Further, the ability of 16K hPRL to prevent angiogenesis in a mouse model of retinopathy (Raisler, B. J., et al. Proc Natl Acad Sci USA 99:8909-14. (2002)) was evaluated. Results indicate that 16K hPRL produced in situ by adenovirus-mediated gene transfer inhibits vascular growth in the mouse retina (Pan et al., Invest Opthalmol V is Sci 45(7): 2413-2419 (2004). Taken together, these results suggested that 16K hPRL may be used as an agent for antitumoral therapy which is performing its effects through the destruction of newly formed blood vessels essential for tumor growth.
The N-terminal 16K fragments of others members of the human prolactin/growth hormone family (PRL/GH family), i.e. 16K growth hormone (16K hGH), 16K growth hormone variant (16K hGH-V) and 16K placental lactogen (16K hPL), were shown also to inhibit angiogenesis while, in contrast, the full-length hormones stimulate capillary formation (Struman, I., et al. Proc Natl Acad Sci U S A 96:1246-51 (1999)). Thus it appeared important to identify, within the 16K fragments, the region that is responsible for their antiangiogenic activity and to understand why these regions are inactive when they are included within the full-length proteins.
The disadvantage of the polypeptides of the state of art was that longer polypeptides are difficult to produce and difficult to purify. Further problems were due to instability and problems concerning their introduction into pharmaceutical compositions.
The object of the present invention was to identify and to provide the effective peptides having antiangiogenic activity which are easier to produce, purify and to handle. A further object was to provide pharmaceutical compositions comprising such peptides having antiangiogenic activity.